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ISRH-seq: a sensitive and robust methodology for R-loop profiling

ABSTRACT: ISRH-seq: a sensitive and robust methodology for R-loop profiling

PROVIDER: PRJNA971645 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR24517619_1.fastq.gz	Fastqsanger.gz
	SRR24517619_2.fastq.gz	Fastqsanger.gz
	SRR24517620_1.fastq.gz	Fastqsanger.gz
	SRR24517620_2.fastq.gz	Fastqsanger.gz
	SRR24517621_1.fastq.gz	Fastqsanger.gz

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ISRH-seq: a sensitive and robust methodology for R-loop profiling

Project description:R-loops serve as key modulators in various biological processes in humans and plants.Current high-throughput methodologies for R-loop profiling have their own intrinsic pros and cons. Therefore, any robust, sensitive, and amenable methods, complementary and/or advantageous to existing methodologies, are much needed for advancing R-loop biology. We here present an RNase H based cleavage combined with DNA polymerase-mediated in situ biotin-dATP/dCTP labeling of newly synthesized DNA for sequencing, termed ISRH-seq, an antibody-independent approach for strand-specific mapping of R-loops. After conducting a parallel comparison with DRIP-seq, we found that ISRH-seq can identify more R-loop peaks with the same sequencing depth. Moreover, ISRH- and DRIP-specific R-loops had distinct genomic and epigenetic features, exhibited distinct relationships with expression of overlapping genes, and functional divergence. Importantly, we found that ISRH-seq can be scalable down to 250-100ng DNA input, indicative of a robust and sensitive high-throughput methodology. In addition, it can be applied for R-loop profiling under stress conditions. Thus, our study provides a valuable orthogonal methodology for comprehensive profiling of R-loops in plants. It could be easily adapted to other non-plant systems, especially for these biologically important tissues or cell types with limited cell numbers, thereby advancing understanding of R-loop biology.

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Sensitive and robust gene expression changes in fish exposed to estrogen - a microarray approach

Project description:Background Vitellogenin is a well established biomarker for estrogenic exposure in fish. However, effects on gonadal differentiation at concentrations of estrogen not sufficient to give rise to a measurable vitellogenin response suggest that more sensitive biomarkers would be useful. Induction of zona pellucida genes may be more sensitive but their specificities are not as clear. The objective of this study was to find additional sensitive and robust candidate biomarkers of estrogenic exposure. Results Hepatic mRNA expression profiles were characterized in juvenile rainbow trout exposed to a measured concentration of 0.87 and 10ng ethinylestradiol/L using a salmonid cDNA microarray. The higher concentration was used to guide the subsequent identification of generally more subtle responses at the low concentration not sufficient to induce vitellogenin. A meta-analysis was performed with data from the present study and three similar microarray studies using different fish species and platforms. Within the generated list of presumably robust responses, several well-known estrogen-regulated genes were identified. Two genes, confirmed by quantitative RT-PCR (qPCR), fulfilled both the criteria of high sensitivity and robustness; the induction of the genes encoding zona pellucida protein 3 and a nucleoside diphosphate kinase (nm23). Conclusions The cross-species, cross-platform meta-analysis correctly identified several robust responses. This adds confidence to our approach used for identifying candidate biomarkers. Specifically, we propose that analyses of nm23 together with zona pellucida genes may increase the possibilities to detect an exposure to low levels of estrogenic compounds in fish.

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Project description:Investigations of 5-hydroxymethylcytosine (5hmC) in biologically and clinically samples and models with low cell numbers have been hampered by the low sensitivity and reproducibility using current 5hmC mapping approaches. Here, we develop a selective 5hmC chemical labeling approach using tagmentation-based library preparation in order to profile nanogram levels of 5hmC isolated from ~1,000 cells (nano-hmC-Seal). Using this technology, we profiled the dynamics of 5hmC across different stages of mouse hematopoietic differentiation. Additionally, applying nano-hmC-Seal to the hematopoietic multipotent progenitor cells in an acute myeloid leukemia (AML) mouse model, we identified leukemia-specific, differentially hydroxymethylated regions that harbor previously reported and as-yet-unidentified functionally relevant factors. The change of 5hmC patterns in AML strongly correlates with the altered gene expression on a global scale. Together, our new approach offers a highly sensitive and robust method to study and detect DNA methylation dynamics from in vivo model and clinical samples.

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